ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response

Abstract

We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.

Figure 1

OxPLs do not influence VEGF mRNA stability and do not up-regulate HIF-1α protein or HIF-1–dependent transcription. (A) HUVECs were incubated in medium 199 containing 2% FCS with or without 130 μM OxPAPC. Actinomycin D was added after 4 hours to stop transcription. After indicated time intervals, the samples were collected and RNA was extracted, reverse transcribed, and analyzed by RT-qPCR for expression of VEGF or COX-2 mRNA. (B) HUVECs were incubated in medium 199 containing 2% FCS with or without 130 μM OxPAPC or 30 mM metal chelator Tiron. At the indicated time points, the cells were scraped into Laemmli sample buffer and analyzed by Western blotting. Staining of the same samples for HIF-1α or EGR-1 proteins is shown. (C) HUVECs grown in 6-well dishes were cotransfected with firefly luciferase promoter-reporter driven by hypoxia response elements and constitutively active Renilla luciferase. After 24 hours, the medium was changed to 2% FCS containing indicated concentrations of OxPAPC (100 μg/mL of OxPAPC = 130 μmol/L). After an overnight incubation, the cells were scraped and activities of both luciferases measured using a dual luciferase assay system. The data represent a ratio of firefly luciferase activity to that of Renilla luciferase. Error bars represent SD.

Figure 2

Lipid structural specificity of OxPL-induced up-regulation of VEGF mRNA. HUVECs were stimulated for 6 hours with indicated lipids resuspended in medium 199 containing 2% FCS. The incubation was terminated by Trizol, followed by RNA extraction and VEGF mRNA quantification by RT-qPCR. The expression levels of VEGF were normalized to those of β₂-microglobulin mRNA. (A) HUVECs were stimulated with 130 μM of OxPAPC or individual OxPLs. (B) HUVECs were treated with increasing concentrations of OxPAPC or lysoPC. (C) The stimulation of HUVECs was performed by increasing concentrations of oxidized arachidonic acid (OxAA). (Inset) Results of HUVEC stimulation with 130 μM of OxPAPC, oxidized (OxAA) or unoxidized arachidonic acid (AA). Error bars represent SD.

Figure 3

Unfolded protein response up-regulates VEGF mRNA. HUVECs were treated for 6 hours in medium 199/2% FCS containing (A) 130 μM OxPAPC, 1 mM dithiothreitol (DTT), 3 μg/mL tunicamycin, 1 μg/mL thapsigargin, or 6 μg/mL brefeldin A, or (B) with increasing concentrations of homocysteine dissolved in medium 199 containing 2% FCS. (C) HUVECs were preincubated overnight with or without 2.5 mM sodium PBA in full growth medium. On the next day, the cells were stimulated for 6 hours with 130 μM OxPAPC or 3 μg/mL tunicamycin in medium 199 containing 2% FCS with or without PBA. After mRNA isolation, levels of mRNA encoding for VEGF were quantified by RT-qPCR. (D) HUVECs were transfected with siRNA against HTJ1 or control siRNA. Forty-eight hours later, the cells were stimulated for 6 hours with 130 μM OxPAPC in medium 199 containing 2% FCS. The incubations were terminated by Trizol, followed by quantification of VEGF mRNA. The data are normalized to the levels of β₂-microglobulin mRNA. Error bars represent SD.

Figure 4

Lipid structural specificity of OxPL-stimulated induction of UPR genes. HUVECs were stimulated with lipids in medium 199 containing 2% FCS. All data obtained by RT-qPCR are normalized to the levels of β₂-microglobulin mRNA. (A) HUVECs were incubated with 130 μM OxPLs containing palmitic and arachidonic acid residues: phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic acid (PA), or phosphatidylserine (PS). Control cells were incubated with the medium containing no lipids. After 4 hours, the cells were scraped into Laemmli buffer and analyzed by Western blotting for ATF4. (B) HUVECs were treated with oxidized or unoxidized phospholipids for 6 hours and then processed for quantification of ATF3 mRNA by RT-qPCR. (C) HUVECs were incubated with individual OxPLs (130 μM each) for 6 hours. (D) HUVECs were incubated for 6 hours with increasing concentrations of OxPAPC or lysoPC. (E) HUVECs were treated with the indicated concentrations of oxidized arachidonic acid (OxAA). (Inset) Data obtained after treatment of HUVECs for 6 hours with 130 μM OxPAPC or oxidized (OxAA) and unoxidized (AA) arachidonic acid. Error bars represent SD.

Figure 5

Induction of VEGF by OxPLs is mediated by the ATF4 branch of UPR. (A,B) Correlation between the expression levels of VEGF and ATF3 (A) or TRB3 (B) in cells treated with various oxidized and unoxidized phospholipids. The data are collected from multiple experiments using different concentrations and molecular species of phospholipids. In all cases, the incubation was performed for 6 hours. Experiments using inhibitors or siRNA treatment were excluded. (C) HUVECs were transfected with siRNA against ATF4. Forty-eight hours later, the cells were stimulated for 6 hours with 130 μM OxPAPC in medium 199 containing 2% FCS. The incubations were terminated by Trizol, followed by quantification of VEGF mRNA. The data are normalized to the levels of β₂-microglobulin mRNA. (D) The experiment was performed as in panel C, but using HAECs. Incubation was performed for 4 hours. (E) HUVECs were pretreated for 30 minutes with 75 μM salubrinal followed by incubation with 130 μM OxPAPC in the presence of salubrinal in medium 199 containing 2% FCS. After 6 hours, the incubation was terminated by Trizol, and the levels of VEGF mRNA were quantified by RT-qPCR. Combined data from 2 independent experiments are shown. (F) HUVECs were incubated with 130 μM OxPAPC in medium 199 containing 2% FCS. After indicated time intervals, the cells were scraped into Laemmli buffer and analyzed by Western blotting for phosphorylated eIF2α. Afterward, the same blots were stained for total eIF2α. Intensity of immunostaining was quantified using chemiluminescent scanner. The normalized signals do not reflect real ratio of phosphoprotein and total protein due to different sensitivities of antibodies and various exposure times. (G) HUVECs were stimulated with OxPAPC (130 μM) resuspended in medium 199 containing 2% FCS. After indicated time intervals, the cells were scraped into Laemmli buffer and analyzed by Western blotting using antibodies against ATF4. (H) HUVECs were treated with OxPAPC (130 μM) or tunicamycin (3 μg/mL) for 3 hours and then processed for ChIP analysis as described in “Chromatin immunoprecipitation.” The figure presents results of ethidium bromide staining of products of PCR amplification of the fragments of VEGF and asparagine synthetase genes. The fragments overlap ATF4-binding sites within the VEGF (“AsnSyn” and “CHOP-1”) and asparagin synthetase (NSRE-1) genes. The antibodies used for ChIP are indicated on the top. The “input” was obtained by amplification of 1% of initial unfractionated cell extract.